The present invention relates to oligonucleotide compositions antisense to bacterial 16S and 23S rRNA and methods for use of such compositions in the treatment of bacterial infection in a mammal.
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Currently, there are several types of antibiotics in use against bacterial pathogens, with a variety of anti-bacterial mechanisms. Beta-lactam antibiotics, such as penicillin and cephalosporin, act to inhibit the final step in peptidoglycan synthesis. Glycopeptide antibiotics, including vancomycin and teichoplanin, inhibit both transglycosylation and transpeptidation of muramyl-pentapeptide, again interfering with peptidoglycan synthesis. Other well-known antibiotics include the quinolones, which inhibit bacterial DNA replication, inhibitors of bacterial RNA polymerase, such as rifampin, and inhibitors of enzymes in the pathway for production of tetrahydrofolate, including the sulfonamides.
Some classes of antibiotics act at the level of protein synthesis. Notable among these are the aminoglycosides, such as kanamycin and gentamycin. These compounds target the bacterial 30S ribosome subunit, preventing the association with the 50S subunit to form functional ribosomes. Tetracyclines, another important class of antibiotics, also target the 30S ribosome subunit, acting by preventing alignment of aminoacylated tRNA""s with the corresponding mRNA codon. Macrolides and lincosamides, another class of antibiotics, inhibit bacterial synthesis by binding to the 50S ribosome subunit, and inhibiting peptide elongation or preventing ribosome translocation.
Despite impressive successes in controlling or eliminating bacterial infections by antibiotics, the widespread use of antibiotics both in human medicine and as a feed supplement in poultry and livestock production has led to drug resistance in many pathogenic bacteria. Antibiotic resistance mechanisms can take a variety of forms. One of the major mechanisms of resistance to beta lactams, particularly in Gram-negative bacteria, is the enzyme beta-lactamase, which renders the antibiotic inactive. Likewise, resistance to aminoglycosides often involves an enzyme capable of inactivating the antibiotic, in this case by adding a phosphoryl, adenyl, or acetyl group. Active efflux of antibiotics is another way that many bacteria develop resistance. Genes encoding efflux proteins, such as the tetA, tetG, tetL, and tetK genes for tetracycline efflux, have been identified. A bacterial target may develop resistance by altering the target of the drug. For example, the so-called penicillin binding proteins (PBPs) in many beta-lactam resistant bacteria are altered to inhibit the critical antibiotic binding to the target protein. Resistance to tetracycline may involve, in addition to enhanced efflux, the appearance of cytoplasmic proteins capable of competing with ribosomes for binding to the antibiotic. Where the antibiotic acts by inhibiting a bacterial enzyme, such as for sulfonamides, point mutations in the target enzyme may confer resistance.
The appearance of antibiotic resistance in many pathogenic bacteria, in many cases involving multi-drug resistance, has raised the specter of a pre-antibiotic era in which many bacterial pathogens are simply untreatable by medical intervention. There are two main factors that could contribute to this scenario. The first is the rapid spread of resistance and multi-resistance genes across bacterial strains, species, and genera by conjugative elements, the most important of which are self-transmissible plasmids. The second factor is a lack of current research efforts to find new types of antibiotics, due in part to the perceived investment in time and money needed to find new antibiotic agents and bring them through clinical trials, a process that may require a 20-year research effort in some cases.
In addressing the second of these factors, some drug-discovery approaches that may accelerate the search for new antibiotics have been proposed. For example, efforts to screen for and identify new antibiotic compounds by high-throughput screening have been reported, but to date no important lead compounds have been discovered by this route.
Several approaches that involve antisense agents designed to block the expression of bacterial resistance genes or to target cellular RNA targets, such as the rRNA in the 30S ribosomal subunit, have been proposed (Good et al., 1998; Rahman et al., 1991). In general, these approaches have been marginally successful, presumably because of poor uptake of the antisense agent (e.g., Summerton et al., 1997), or the requirement that the treated cells show high permeability for antibiotics (Good et al., 1998).
There is thus a growing need for new antibiotics that (i) are not subject to the principal types of antibiotic resistance currently hampering antibiotic treatment of bacteria, (ii) can be developed rapidly and with some reasonable degree of predictability as to target-bacteria specificity, (iii) can also be designed for broad-spectrum activity, (iv) are effective at low doses, meaning, in part, that they are efficiently taken up by wild-type bacteria or even bacteria that have reduced permeability for antibiotics, and (v) show few side effects.
In one aspect, the invention provides an antibacterial compound, consisting of a substantially uncharged antisense oligomer containing from 8 to 40 nucleotide subunits, including a targeting nucleic acid sequence at least 10 nucleotides in length which is complementary to a bacterial 16S or 23S rRNA nucleic acid sequence. Each of the subunits comprises a 5- or 6-membered ring supporting a base-pairing moiety effective to bind by Watson-Crick base pairing to a respective nucleotide base in the bacterial nucleic acid sequence. Adjacent subunits are joined by uncharged linkages selected from the group consisting of: uncharged phosphoramidate, phosphorodiamidate, carbonate, carbamate, amide, phosphotriester, alkyl phosphonate, siloxane, sulfone, sulfonamide, sulfamate, thioformacetyl, and methylene-N-methylhydroxylamino, or by charged linkages selected from the group consisting of phosphate, charged phosphoramidate and phosphorothioate. The ratio of uncharged linkages to charged linkages in the oligomer is at least 4:1, preferably at least 5:1, and more preferably at least 8:1. In one embodiment, the oligomer is fully uncharged.
Preferably, the oligomer is able to hybridize with the bacterial sequence at a Tm substantially greater than the Tm of a duplex composed of a corresponding DNA and the same bacterial sequence. Alternatively, the oligomer is able to hybridize with the bacterial sequence at a Tm substantially greater than 37xc2x0 C., preferably greater than 50xc2x0 C., and more preferably in the range of 60-80xc2x0 C.
In one embodiment, the oligomer is a morpholino oligomer. The uncharged linkages, and, in one embodiment, all of the linkages, in such an oligomer are preferably selected from the group consisting of the structures presented in FIGS. 2A through 2D. Particularly preferred are phosphorodiamidate-linked oligomers, as represented at FIG. 2B, where Xxe2x95x90NR2, R being hydrogen or methyl, Yxe2x95x90O, and Zxe2x95x90O.
The length of the oligomer is preferably 12 to 25 subunits. In one embodiment, the oligomer is a phosphorodiamidate-linked morpholino oligomer having a length of 15 to 20 subunits, and more preferably 17-18 subunits.
In selected embodiments, the targeting sequence is a broad spectrum sequence selected from the group consisting of SEQ ID NOS:15, 16, and 21-25. In other embodiments, the targeting sequence is complementary to a Gram-positive bacterial 16S rRNA consensus sequence, e.g., SEQ ID NOS:27-28, or is complementary to a Gram-negative bacterial 16S rRNA consensus sequence, e.g. SEQ ID NOS:29-30.
Other targeting sequences can be used for treatment of an infection produced by various organisms, for example:
(a) E. coli, where the sequence is selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:35;
(b) Salmonella thyphimurium, where the sequence is selected from the group consisting of SEQ ID NO:18 and SEQ ID NO:36;
(c) Pseudomonas aeruginosa, where the sequence is selected from the group consisting of SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42 and SEQ ID NO:43;
(d) Vibrio cholera, where the sequence is selected from the group consisting of SEQ ID NO:45, SEQ ID NO:46 and SEQ ID NO:47;
(e) Neisseria gonorrhoea, where the sequence is selected from the group consisting of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51;
(f) Staphylococcus aureus, where the sequence is selected from the group consisting of SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:55;
(g) Mycobacterium tuberculosis, where the sequence is selected from the group consisting of SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59;
(h) Helicobacter pylori, where the sequence is selected from the group consisting of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62 and SEQ ID NO:63;
(i) Streptococcus pneumoniae, where the sequence is selected from the group consisting of SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66 and SEQ ID NO:67;
(j) Treponema palladium, where the sequence is selected from the group consisting of SEQ ID NO:69, SEQ ID NO:70 and SEQ ID NO:71;
(k) Chlamydia trachomatis, where the sequence is selected from the group consisting of SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:75;
(l) Bartonella henselae, where the sequence is selected from the group consisting of SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78 and SEQ ID NO:79;
(m) Hemophilis influenza, where the sequence is selected from the group consisting of SEQ ID NO:81, SEQ ID NO:82 and SEQ ID NO:83;
(n) Shigella dysenterae, where the sequence is presented as SEQ ID NO:88; or
(o) Enterococcus faecium, where the sequence is presented as SEQ ID NO:92.
In other embodiments, the targeting sequence is an antisense oligomer sequence selected from one of the following groups, for use in treatment of an infection produced by:
(a) E. coli, Salmonella thyphimurium and Shigella dysenterae, where the sequence is selected from the group consisting of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:86 and SEQ ID NO:87;
(b) E. coli, Salmonella thyphimurium and Hemophilis influenza, where the sequence is presented as SEQ ID NO:31;
(c) E. coli and Shigella dysenterae, where the sequence is presented as SEQ ID NO:17;
(d) E. coli, Salmonella thyphimurium, Shigella dysenterae, Hemophilis influenza and Vibrio cholera, where the sequence is presented as SEQ ID NO:44;
(e) Staphylococcus aureus and Bartonella henselae, where the sequence is presented as SEQ ID NO:52;
(f) Salmonella thyphimurium, Hemophilis influenza and Treponema palladium, where the sequence is presented as SEQ ID NO:68; or
(g) E. coli, Salmonella thyphimurium, Shigella dysenterae, Hemophilis influenza and Neisseria gonorrhoea, where the sequence is presented as SEQ ID NO:84.
In a related aspect, the invention provides a method of treating a bacterial infection in a human or mammalian animal subject, by administering to the subject, in a pharmaceutically effective amount, a substantially uncharged antisense oligomer as described above. Various selected embodiments of the oligomer and the target sequence are as described above. Preferably, the antisense oligomer is administered in an amount and manner effective to result in a peak blood concentration of at least 200-400 nM antisense oligomer. The method can be used, for example, for treating bacterial infections of the skin, wherein administration is by a topical route, or for use in treating a bacterial respiratory infection, wherein administration is by inhalation.
In a further related aspect, the invention provides a livestock and poultry food composition containing a food grain supplemented with a subtherapeutic amount of an antibacterial compound, said compound consisting of a substantially uncharged antisense oligomer as described above.
Also contemplated is, in a method of feeding livestock and poultry with a food grain supplemented with subtherapeutic levels of an antibiotic, an improvement in which the food grain is supplemented with a subtherapeutic amount of an antibacterial compound of the type described above.
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples.